Heart failure (HF) continues to be a major health problem in the U.S. There is a great need to understand mechanisms of cardiac dysfunction as well as begin to explore potential novel therapeutic targets and therapies. Previously, we have investigated the role of beta-adrenergic receptor (betaAR) signaling in the hypertrophic and failing heart and found that the betaAR kinase (betaARK1 or GRK2) plays a critical role in the pathogenesis of HF. Moreover, we have found that expression of a peptide inhibitor (betaARKct) of betaARK1 can lead to profound beneficial effects on cardiac function and appears to be a novel therapeutic strategy for HF. In addition to betaR signaling, it is well known that calcium (Ca2+) handling and sarcoplasmic reticulum (SR) function in the failing cardiomyocyte is deranged and transgenes targeting various aspects of this cellular process can have salutary effects on cardiac function. We have recently found that the Ca2+ binding protein S100A1 is a novel regulator of myocardial contractility. Through the use of myocardial-targeted S100A1 transgenic mice and in vitro adenoviral-mediated gene transfer to myocytes, S100A1 overexpression can enhance cardiac contractility and SR Ca2+ uptake. This proposal is formulated to test whether S100A1 gene addition can correct SR Ca2+ signaling abnormalities in the failing heart in vivo and how it may compare to the betaARKct including pre-clinical testing of viral-mediated HF gene therapy. These studies will also lead to novel molecular mechanistic insight in the role of this Ca2+ binding protein in cardiac regulation and how it may relate to betaAR signaling. The Central Hypothesis of this proposal is that altered expression of S100A1 in HF contributes to myocyte Ca2+-signaling abnormalities and cardiac dysfunction and that myocardial gene transfer of S100A1 represents, in addition to the betaARKct, a novel therapeutic strategy for HF. Specific Aims are: (1) To utilize novel mouse models to determine the in vivo role of S100A1 in failing myocardium. (2) To determine the role of the betaAR system in S100A1 function in the heart through the use of novel mouse models. (3) To investigate the potential therapeutic role and mechanism of action of S100A1 in HF by utilizing intracoronary, myocardial gene delivery to HF rabbits. (4) To establish conditions for efficient in vivo intracoronary delivery of advanced adenoviral and AAV vectors to a large animal (pig) and to test, in a preclinical manner, betaARKct and S100A1 gene delivery to a pacing-induced HF model in the pig.